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WO2014036167A1 - Détection de variants dans des données de séquençage et un étalonnage - Google Patents

Détection de variants dans des données de séquençage et un étalonnage Download PDF

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Publication number
WO2014036167A1
WO2014036167A1 PCT/US2013/057128 US2013057128W WO2014036167A1 WO 2014036167 A1 WO2014036167 A1 WO 2014036167A1 US 2013057128 W US2013057128 W US 2013057128W WO 2014036167 A1 WO2014036167 A1 WO 2014036167A1
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Prior art keywords
sequencing
data
variants
tumor
filters
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PCT/US2013/057128
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English (en)
Inventor
Kristian Cibulskis
Gad Getz
Michael Lawrence
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The Broad Institute, Inc.
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Priority to EP13832861.2A priority Critical patent/EP2891099A4/fr
Publication of WO2014036167A1 publication Critical patent/WO2014036167A1/fr
Priority to US14/633,321 priority patent/US20150178445A1/en

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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/10—Ploidy or copy number detection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/10—Sequence alignment; Homology search

Definitions

  • HHSN261201000055C awarded by the Department of Health and Human Services
  • TECHNICAL FIELD [0003] This disclosure relates generally to sequencing data processing and benchmarking, and in particular, to detecting variants in sequencing data.
  • Cancer is a disease of the genome wherein somatic genetic alterations transform normal cells into malignant cells. Detecting, cataloguing and interpreting these somatic events are at the core of a rapidly increasing number of cancer genome projects such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC), which involve thousands of cases harboring millions of mutations. As sequencing moves from research into clinical use, for example, as a tool for diagnostic, even more cases will need to be characterized.
  • TCGA Cancer Genome Atlas
  • ICGC International Cancer Genome Consortium
  • Somatic single-nucleotide substitutions are an important and common mechanism for altering gene function in cancer. Yet, they are challenging to identify. First, they occur at a very low frequency in the genome, ranging from 0.1 to 100 mutations per megabase, depending on tumor type. Second, the alterations may be present only in a small fraction of the DNA molecules originating from the specific genomic locus. The reasons include contamination of cancer cells with surrounding stromal cells; local copy-number variation within the cancer genome; and presence of a mutation within only a sub-population of the tumor cells ('subclonality'). The fraction of DNA harboring an alteration ('allelic fraction') has been reported to be as low as 0.05 for highly impure tumors. Consequently, a mutation calling method must be highly sensitive to somatic mutations with very low allelic fractions (i.e. fraction of sequencing reads that support the mutation).
  • the sensitivity and specificity of any somatic mutation caller varies along the genome. They depend on factors including, for example, depth of sequence coverage in the tumor and normal; the local sequencing error rate; the allelic fraction of the mutation; and the evidence thresholds used to declare a mutation. Understanding how sensitivity and specificity depend on these factors is necessary for designing experiments with adequate power to detect mutations at a given allelic fraction, as well as for inferring the mutation frequency along the genome, which is a key parameter for understanding mutational processes and significance analysis.
  • the current subject matter relates to a computer-implemented method.
  • the method can include receiving aligned sequencing data; applying one or more filters to the aligned sequencing data; using the filtered data as input, applying a first classifier to determine if any alteration is present beyond an expected threshold due to a sequencing error and identifying one or more candidate variants; passing the one or more identified candidate variants through one or more additional filters to remove one or more false positives; and determining a somatic status of the one or more filtered candidate variants using a second classifier.
  • At least one of the above can be performed on at least one processor.
  • the sequencing data may include DNA sequencing or RNA sequencing data.
  • the one or more variants are mutations, point mutations, somatic point mutations, or germline point mutations.
  • the one or more false positives are created by correlated sequencing noise.
  • a Panel of Normals is used to identify one or more false positives.
  • At least one of the first and second classifiers can be a Bayesian classifier.
  • the one or more filters include a proximal gap filter which rejects variants with neighboring insertion and/or deletion events. In some implementations, the one or more filters include a poor mapping region filter which rejects sites having a determined mapping quality score of zero. In some
  • the one or more filters include a clustered position filter which looks for correlation in the position of mutant alleles within their reads.
  • the one or more filters include a strand bias filter which rejects sites where a distribution of strand observations of mutant allele is biased compared to the allele of the reference genome.
  • the one or more filters include a tri allelic site filter which excludes sites each having at least three alleles beyond what is expected by sequencing error.
  • the one or more filters include an observed in control filter which uses sequencing data from a matched normal as control data to eliminate sites where the reference genome has evidence of mutant allele.
  • a system for detecting one or more variants from sequencing data can include means for receiving aligned sequencing data; means for applying one or more filters to the aligned sequencing data; means for using the filtered data as input, applying a first classifier to determine if any alteration is present beyond an expected threshold due to a sequencing error and identifying one or more candidate variants; means for passing the one or more identified candidate variants through one or more additional filters to remove one or more false positives; and means for determining a somatic status of the one or more filtered candidate variants using a second classifier.
  • a method for benchmarking performance of variant detection includes providing variants that were discovered in deep- coverage data sets; down-sampling by randomly excluding a subset of reads of the data set at sites of known validated variants; repeating the down-sampling one or more times and estimating a sensitivity as a fraction of the times the known variants are detected. At least one of the above is performed by at least one data processor.
  • a method for benchmarking performance of variant detection includes creating a normal virtual tumor that has no true variants; providing sequence data from a single normal sample; assigning reads of the sequence data to be either "tumor” or "normal” to a desired depth; and measuring specificity by comparing the normal virtual tumor against the sequence data. At least one of the above is performed by at least one data processor.
  • Articles of manufacture are also described that comprise computer executable instructions permanently stored on non-transitory computer readable media, which, when executed by a computer, causes the computer to perform operations herein.
  • computer systems are also described that may include a processor and a memory coupled to the processor. The memory may temporarily or permanently store one or more programs that cause the processor to perform one or more of the operations described herein.
  • operations specified by methods described herein can be implemented by one or more data processors either within a single computing system or distributed among two or more computing systems.
  • the present subject matter provides a novel somatic point mutation caller, which we call "MuTect,” is believed to be superior to prior methods in terms of sensitivity, particularly for low allelic fraction events, while remaining highly specific. This uniquely positions the method to deeply explore the mutational landscape of highly impure tumor samples, as well as the subclones with a tumor. The ability to characterize these subclonal events is not only critical to understanding tumor evolution both in disease progression and response to treatment, but also as a clinical diagnostic for personalized cancer therapy.
  • a differentiator of the current subject matter allowing it to be sensitive to low allele fraction mutations is the explicit modeling of alternate alleles at any frequency, whereas alternative methods typically assume heterozygous genotypes as the basis for their calculations.
  • Down-sampling This approach involves studying somatic mutations that were discovered in deep-coverage cancer data sets and then experimentally validated, to see if these "gold-standard" mutations would have been found with lower coverage. Down-sampling can be accomplished, for example, by randomly excluding a subset of the reads at the sites of these validated mutations. For depths of coverage from 5x to 50x in the tumor and normal, the down-sampling procedure can be performed repeatedly and the sensitivity can be estimated as the fraction of times the known mutation is detected. Notably, down-sampling preserves the expected allelic fraction of the original mutation, because reads are removed regardless whether or not they contain the alternate allele.
  • a virtual tumor can be created that has no true mutations. Using sequence data from a single normal sample, the reads can be assigned to be either 'tumor' or 'normal' to a desired depth. By applying methods to this virtual tumor-normal pair, the specificity of the method can be easily measured because any somatic mutations identified are necessarily false positives.
  • a virtual tumor can be created that has true mutations only at known sites.
  • mutations can be introduced by substituting reads from a second normal sample ("B").
  • B normal sample
  • sites at which B contains heterozygous germline variants not found in A can be identified.
  • Reads in the virtual tumor with variant-containing reads from B can be replaced, following a binomial distribution given a specified allelic fraction.
  • One advantage of using germline events is that they are frequent ( ⁇ 1000/Mb) and accurately detected, as they have often been genotyped by multiple technologies. In this manner, real sequencing data can be used to introduce somatic mutations within a virtual tumor to any desired depth and allelic fraction.
  • the two benchmarking approaches can be complementary: down- sampling uses real somatic mutations but is limited to previously detected and validated mutations, whereas the virtual tumor approach can generate a large datasets but reflects the distribution of events that occur in the germline.
  • Figure 1 is a process flow diagram illustrating an exemplary implementation of the present subject matter
  • Figures 2a and 2b show sensitivity and specificity of results in accordance with some implementations of the present subject matter
  • Figures 3a-3f show various results of specificity of somatic classification and variant detection using an exemplary implementation of the present subject matter
  • Figures 4a-4d show comparisons of various benchmarks of implementations of the present subject matter against different detection methods.
  • Figure 5 is a process flow diagram illustrating an exemplary implementation of the present subject matter.
  • the present subject matter is directed to the detection of variants, which include, for example, alterations, allelic variants, mutations and polymorphisms.
  • the sequencing data may include, for example, DNA, RNA, cDNA, and/or other genetic sequencing data.
  • down-sampling can use subsets of reads from primary sequencing data of validated somatic mutations to measure the sensitivity with which a mutation caller identifies the known mutations.
  • Subsets can be generated by randomly excluding reads from the experimentally-derived data set until a desired depth of coverage is reached.
  • down-sampling can preserve the expected allelic fraction of the original mutation because reads are removed regardless whether or not they contain the mutant allele.
  • the down-sampling approach can potentially be limited in four respects: (i) the number of validated events is typically small, resulting in larger error bars for the sensitivity estimate; (ii) because allele fractions are preserved, only previously validated allele fractions can be explored; (iii) the analysis excludes any mutations that were not originally detected and hence may overestimate the true sensitivity; and (iv) specificity cannot be measured.
  • virtual tumors and normal can be created, at controlled depths, from sequencing data generated by two different sequencing experiments of the same normal sample (designated A). All mutations identified are necessarily false positives.
  • somatic mutations can be simulated at controlled allele fractions by replacing selected reads in the virtual tumor with reads from a second sample (designated B) at loci where sample A is reference and sample B harbors a high confidence germline heterozygous event. The ability of an algorithm to detect these simulated somatic mutations can then be assessed. In this manner, sensitivity can be measured using real sequencing data at a desired depth of coverage and allelic fraction.
  • the two benchmarking approaches can be complementary. Down-sampling can use real somatic mutations, but can be limited in the parameter regimes it can explore, and it cannot measure specificity directly.
  • the virtual tumor approach does not have these limitations. However, it simulates somatic mutations using germline events, which differ from somatic mutations in their nucleotide substitution frequencies and context. As recalibrated base qualities vary for the different bases (owing to biases in machine errors), there is variable sensitivity to detect different substitutions (Fig. 2). Because the difference in sensitivity is minimal, all the germline events can be chosen. However, it is possible with the virtual tumor approach to simulate the mutation spectrum of a specific tumor type by reweighting the germline events to match the expected mutation spectrum of the tumor.
  • the present subject matter takes as input sequence data from matched tumor and normal DNA, following alignment of the data to a reference genome and standard preprocessing steps. Examples of the preprocessing steps can be found in DePristo, M.A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nature genetics 43, 491-498 (201 1), the contents of which are incorporated herein by reference. In some implementations, the present subject matter operates on each genomic locus
  • the process flow 500 includes receiving DNA (e.g.) sequencing data at 502, aligning DNA sequencing data to a reference genome at 504, applying one or more filters to the aligned DNA sequencing data at 506, applying a first Bayesian classifier on the filtered data and identifying one or more candidate mutations at 508, applying one or more additional filters to the candidate mutation(s) at 510, and applying a second Bayesian classifier on the filtered candidate mutation(s) and determining a variant (somatic) status or classification of the filtered candidate mutation(s) at 512.
  • the present subject matter can take as input paired tumor and normal next generation sequencing- data and, after removing low quality reads, determines if there is evidence for a variant beyond the expected random sequencing errors (variant detection will be discussed in more detail below).
  • Candidate variant sites are then passed through, for example, one or more (including all) filters to remove sequencing and alignment artifacts:
  • Triallelic Site filter excludes sites where there appear to be at least three alleles at the site beyond what is expected by sequencing error suggesting a site not accurately modeled by the base quality scores;
  • a Panel of Normals can be used to screen out remaining false positives caused by rare error modes only detectable using more samples. Finally, the somatic or germline status of passing variants is determined using the matched normal.
  • the present subject matter can take as input sequence data from matched tumor and normal DNA after alignment of the reads to a reference genome and preprocessing steps discussed above, which include, for example, marking of duplicate reads, recalibration of base quality scores and local realignment.
  • the method operates on each genomic locus independently and consists of four key steps (Fig. 1 ): (i) Removal of low-quality sequence data (based on known methods); (ii) variant detection in the tumor using a Bayesian classifier; (iii) filtering to remove false positives resulting from correlated sequencing artifacts that are not captured by the error model; and (iv) designation of the variants as somatic or germline by a second Bayesian classifier.
  • variants in the tumor can be identified by analyzing the data at each site under, for example, two alternative models:
  • a variant model M 1 which assumes the site contains a true variant allele m at allele fraction / and also allows, as in Mo, for the possibility of sequencing errors.
  • the allele fraction / is unknown and is estimated as the fraction of tumor reads that support m.
  • m can be declared to be a candidate variant if the log-likelihood ratio of the data under the variant and reference models - that is, the LOD score (log odds) - exceeds a predefined decision threshold that depends on the expected mutation frequency and the desired false positive rate (Online Methods).
  • ROC Receiver Operating Characteristic
  • the LOD score is useful as a threshold for declaring the presence of mutations, as can be observed in the concordance of predicted sensitivity and measured sensitivity from the virtual tumor approach ( Figure 2a, solid grey line vs. dashed line). Nonetheless, the LOD score cannot be immediately translated into the probability that a variant is due to true mutation rather than to sequencing error because the LOD score is calculated under an assumption of independent sequencing errors and accurate read placement. As will be discussed below, these assumptions are incorrect and as a result, although direction application of the LOD score accurately estimates the sensitivity to detect a mutation, it can substantially underestimate the true false positive rate.
  • Figures 2a and 2b show the sensitivity as a function of sequencing depth and allelic fraction.
  • Results using a model of independent sequencing errors with uniform Q35 base quality scores and accurate read placement (solid grey) are shown as well as results from the virtual tumor approach for the standard (STD, dashed green) and high-confidence (HC, solid green) configuration.
  • a typical setting of ⁇ 6.3 is marked with black dots.
  • the calculated sensitivity using a model of independent sequencing errors and accurate read placement with uniform Q35 ase quality scores (solid lines) are shown as well as results from the virtual tumor approach (circles) and the downsampling of validated colorectal mutations (diamonds). Error bars represent 95% CIs.
  • the sensitivity of the method is similar as estimated by the calculation and the virtual tumor benchmark both with (HC) and without (STD) filters. This demonstrates that the model is accurate with respect to detection and that the filters do not adversely impact sensitivity.
  • each variant detected in the tumor is designated as somatic (not present in the matched normal), germline (present in the matched normal) or variant (present in the tumor, but indeterminate status in the matched normal due to insufficient data).
  • a LOD score can be used that compares the likelihood of the data under models in which the variant is present (at 50% frequency) or absent in the matched normal (Online Methods).
  • the power to make a germline classification given the data and threshold can be calculated.
  • insufficient data for classification is declared if there is less than 95% power.
  • public germline variation databases can be used as a prior probability of an event being germline. Sensitivity
  • these benchmarking methods can be further applied to further evaluate the sensitivity of our mutation detection method, with the different filtering options (STD, HC and HC+PON), to detect mutations as a function of sequencing depth and allelic fraction (Figure 2b).
  • the sensitivity can be calculated under a model of independent sequencing errors and accurate read placement using, for example, a statistical test given an allelic fraction; tumor sequencing depth; and assuming all bases have a fixed base quality score of Q35 (approximate mean base quality score in simulation data; Online Methods).
  • HC+PON may not be used in the virtual tumor sensitivity benchmark because it discards common germline sites.
  • the present subject matter is a highly sensitive detection method. It can detect mutations, for example, at a site with 3 Ox depth in the tumor (typical of whole genome sequencing) and an allele fraction of 0.2 with 95.6% sensitivity.
  • the sensitivity can be increased to 99.9% by sequencing deeper (e.g., to a depth of 50x), and drops to, e.g., 58.9% for detecting mutations with allelic fraction of 0.1 (at 30x) ( Figure 2b).
  • the present subject matter can have, e.g., 66% sensitivity for 3% allele fraction events. It is this sensitivity to detect low-allele fraction events that uniquely positions the present subject matter to analyze samples with low purity or with complex subclonal structure.
  • the virtual tumor approach can be used, for example, across 1 Gb of NA12878 at various depths in the virtual tumor and at 3 Ox in the virtual normal. All detected events are false positives, but to eliminate those due to under-calling germline events from consideration, we excluded all known germline variant sites.
  • STD no filters
  • the false positive rate increased with depth (from 6.7/mb at 5x to 20.1/mb at 30x) (Fig. 3a). This is due to the increased power to call mutations with lower allele fractions, which are enriched with false positives (Fig. 3b).
  • the HC filters reduce the false positive rate by an order of magnitude (1.00/mb at 30x).
  • the Panel of Normals filters out remaining rare, but recurrent, artifacts (0.51/mb at 30x).
  • Certain filters such as the Poor Mapping filter, have the biggest effect at low depths whereas other filters are more invariant to depth, such as the Proximal Gap filter (Fig. 3c).
  • the Clustered Position filter rejects the most sites exclusively. However, the majority of false positives are rejected by several filters.
  • the filters specifically address these additional errors and reduce the false positive rate by an order of magnitude (from 21.3/mb to 0.90/mb at 30x tumor depth).
  • the Panel of Normals (HC+PON) then filters out remaining rare, but recurrent, artifacts. Certain filters, such as the Poor Mapping filter, have the biggest effect at low depths whereas other filters are more invariant to depth, such as the Proximal Gap filter ( Figure 3 c).
  • the Clustered Position filter rejects the most sites exclusively, although multiple filters reject the majority of false positives.
  • false positives can be further reduced by taking each read in the tumor and normal, and realigning them to a reference genome with stringent alignment settings.
  • the resulting alignments can be re-processed by the present subject matter to see if enough evidence for the mutation exists after considering the more stringent alignments.
  • Figures 4a-4d show the benchmarking mutation detection methods. Specifically, the sensitivity of the methods was evaluated with regard to allele fraction and tumor sequencing depth using the virtual tumor (Fig. 4a) and down-sampling approaches, and a sharp distinction in sensitivity was observed, particularly at lower allele fractions. Data were analyzed for 3 Ox sequence coverage. In the standard configurations, all methods show > 99.3% sensitivity for mutations at an allele fraction of 0.4. However, in the HC configurations, the present subject matter, JointSNVMix and Strelka remain sensitive, 98.8%), 96.6% and 98.5% respectively, whereas SomaticSniper drops to 91.5%.
  • the present subject matter HC can detect more than half of the mutations (53.2%), whereas Strelka HC detects only 29.7%), JointSNVMix HC drops to 16.8% and SomaticSniper HC falls to 7.4%.
  • the present subject matter HC has 16.0% sensitivity but can be increased to 51.9%) with 60x coverage.
  • SomaticSniper HC have a sensitivity of ⁇ 2.0%, and the sensitivity does not increase appreciably with tumor sequencing depth. Strelka HC detects just 4.6%> of the events at 30x and only increases to 20.8% at 60x. Sensitivity for such low allelic fraction events is critical for characterizing impure tumors or subclonal mutations in heterogeneous tumors, and it appears that the present subject matter is much more sensitive in this regime.
  • cancer genome community will greatly benefit from a systematic performance measurement using the approaches described here across the entire parameter space of tumor and normal depths and mutation allele fraction.
  • the approaches described herein can also be extended in the future to other alterations such as indels or rearrangements.
  • the cancer genome community is eager to adopt new and improved methods but require detailed
  • the present subject matter is shown to be much more sensitive at a given specificity than competing methods, allowing one to more comprehensively characterize the landscape of somatic mutations, particularly those present in a small fraction of cancer cells. Moreover, this can be done with standard sequencing depths enabling analysis of the large datasets that are being generated worldwide. Analysis of subclonal mutations and changes in the fractions of cancer cells which harbor them is a powerful way to study the evolution of subclones as they progress during treatment, metastasis and relapse. In particular, we demonstrated that the presence of subclonal mutations in genes involved in driving chronic lymphocytic leukemia (CLL) is an independent prognostic factor beyond the currently used clinical parameters.
  • CLL chronic lymphocytic leukemia
  • Figure 1 is an overview of somatic point mutation detection using the present subject matter.
  • the present subject matter takes as input tumor (T) and normal (N) next generation sequencing data and, after removing low quality reads, determines if there is evidence for a variant beyond the expected random sequencing errors.
  • Candidate variant sites are then passed through six filters to remove artifacts (Table 1).
  • a Panel of Normals can be used to screen out remaining false positives caused by rare error modes only detectable in additional samples.
  • somatic or germline status of passing variants is determined using the matched normal.
  • Figure 2 shows sensitivity as a function of sequencing depth and allelic fraction.
  • Figure 3 shows specificity of variant detection and variant classification using virtual tumor approach, (a) Somatic miscall error rate for true reference sites as a function of tumor sequencing depth for the STD (red), HC (blue) and HC+PON (green) configurations of the present subject matter. Error bars represent 95% CIs. (b) Distribution of allele fraction for all miscalls as a function of tumor sequencing depth, (c) Fraction of events rejected by each filter; hashed regions indicate events rejected exclusively by each filter, (d) Somatic miscall error rate for true germline SNP sites by sequencing depth in the normal when the site is known to be variant in the population (blue) and novel (red). Error bars represent 95%> CIs. (e,f) Mean power as a function of sequencing depth in the normal to have classified these events as germline or somatic at novel germline sites (e) and known germline variant sites (f).
  • Figure 4 shows benchmarking mutation detection methods
  • Reads are preprocessed differently according to how they will be used: detection of the variant in the tumor, discovery of an artifact in the normal or for somatic classification.
  • This filter attempts to remove false positives caused by nearby misaligned small insertion and deletion events.
  • the site can be rejected if there are > 3 reads with insertions within an 1 lbp window centered on the candidate mutation OR if there are > 3 reads with deletions within the same 1 lbp window.
  • This filter attempts to remove false positives caused by sequence similarity in the genome by looking at the fraction of reads which have a mapping quality score of zero. Candidates are rejected of > 50% of the reads in the tumor and normal have a mapping quality of zero.
  • This filter attempts to reject false positives caused by calling triallelic sites where the normal is heterozygous with alleles A/B and the present subject matter is considering an alteration of allele C. Although this is biologically possible, and remains an area for future improvement in mutation detection, calling at these sites generates many false positives and therefore they are currently filtered out by default.
  • This filter attempts to reject false positives caused by context specific sequencing errors where the vast majority of the alternate alleles are observed in a single direction of reads. In some implementations, this test is performed by stratifying the reads by direction and then performing the core detection statistic on the data.
  • the method calculates the median and median absolute deviation of the distance from both the start and end of the read and reject sites that have a median ⁇ 10 (near the start/end of the alignment) and a median absolute deviation ⁇ 3 (clustered).
  • a panel of normal samples can be employed as a screen.
  • the present subject matter can run on them as if they were tumors without matched normal and all artifact- processing disabled ( ⁇ artifact_detection_mode). From this data, a VCF file is created for the sites that were identified by the present subject matter in two or more samples.
  • This VCF can then supplied to the caller, which rejects these sites. However, if the site was present in the supplied VCF of known mutations (--cosmic) it is retained because these sites could represent known recurrent somatic mutations which have been detected in the panel of normal when the normal are from adjacent tissue or have some contamination tumor DNA.
  • Variant (Somatic) Classification To perform this classification, we use a similar classifier to the one described above. In this case /, in M 1 , is conservatively set to 0.5 for a germline heterozygous variant.
  • a threshold of 10 can be set, which is higher than the threshold for ⁇ ⁇ so as to obtain more confidence in the somatic classification as misclassified germline events will quickly appear to be significant in downstream somatic analysis due to their elevated population frequency at recurrent sites as compared to real somatic events.
  • the public dbSNP database can be used to make this distinction.
  • the virtual tumor approach begins with a high coverage (60x) whole genome sample sequenced by 1000 Genomes (NA 12878).
  • chromosome 20 is focused, as opposed to the entire genome, for computational efficiency.
  • the first step is to randomly divide the sequencing data in to several partitions.
  • 12 partitions is created from the original 60x data, therefore creating data partitions with ⁇ 5x each.
  • This can be accomplished by sorting the BAM by name using SortSam from the Picard (http://picard.sourceforge.net) tools to effectively give the reads random ordering.
  • Each read can be randomly allocated to one of the partitions and write it to a partition specific BAM file.
  • partitions can be designated as the tumor and others as the normal and process them through the present subject matter. Any somatic mutations identified in this process are false positives as they are either germline events that are mis-sampled in the normal, or erroneous variants due to sequencing noise identified in the partitions designated as tumor. Because the present subject matter can accept multiple BAM files for each the tumor and normal, there is no need to merge the partitions a priori. However, because other methods do not have this capability the individual BAMs can also be merged.
  • NA12891 can be used and sequenced to 60x as part of the 1000 Genomes Project. Using the published high confidence genotypes for those samples from the 1000 Genomes Project, a set of sites that are heterozygous in NA 12891 and homozygous for the reference in NA 12878 can be identified.
  • SomaticSpike can also be used with MuTect to perform a mixing experiment in-silico.
  • this utility attempts to replace a specified fraction of reads drawn from a binomial distribution in the NA12878 data with reads from the NA12891 data therefore simulating a somatic mutation of known location and allele fraction. If there are not enough reads in NA 12891 to replace the desired reads in NA 12878 the site is skipped.
  • the output of this process is a BAM with the in-silico variants and a set of locations of those variants.
  • the sensitivity is then the probability of observing k or more reads given the allelic fraction and depth.
  • aspects of the subject matter described herein can be embodied in systems, apparatus, methods, and/or articles depending on the desired configuration.
  • various implementations of the subject matter described herein can be realized in digital electronic circuitry, integrated circuitry, specially designed application specific integrated circuits (ASICs), computer hardware, firmware, software, and/or combinations thereof.
  • ASICs application specific integrated circuits
  • These various implementations can include implementation in one or more computer programs that are executable and/or interpretable on a programmable system including at least one programmable processor, which can be special or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device, and at least one output device.
  • machine-readable signal refers to any signal used to provide machine instructions and/or data to a programmable processor.
  • the machine-readable medium can store such machine instructions non-transitorily, such as for example as would a non-transient solid state memory or a magnetic hard drive or any equivalent storage medium.
  • the machine- readable medium can alternatively or additionally store such machine instructions in a transient manner, such as for example as would a processor cache or other random access memory associated with one or more physical processor cores.
  • the subject matter described herein can be implemented on a computer having a display device, such as for example a cathode ray tube (CRT) or a liquid crystal display (LCD) monitor for displaying information to the user and a keyboard and a pointing device, such as for example a mouse or a trackball, by which the user may provide input to the computer.
  • a display device such as for example a cathode ray tube (CRT) or a liquid crystal display (LCD) monitor for displaying information to the user and a keyboard and a pointing device, such as for example a mouse or a trackball, by which the user may provide input to the computer.
  • CTR cathode ray tube
  • LCD liquid crystal display
  • a keyboard and a pointing device such as for example a mouse or a trackball
  • Other kinds of devices can be used to provide for interaction with a user as well.
  • feedback provided to the user can be any form of sensory feedback, such as for example visual feedback, auditory feedback, or tactile feedback
  • touch screens or other touch-sensitive devices such as single or multi-point resistive or capacitive trackpads, voice recognition hardware and software, optical scanners, optical pointers, digital image capture devices and associated interpretation software, and the like.
  • the subject matter described herein can be implemented in a computing system that includes a back-end component, such as for example one or more data servers, or that includes a middleware component, such as for example one or more application servers, or that includes a front-end component, such as for example one or more client computers having a graphical user interface or a Web browser through which a user can interact with an implementation of the subject matter described herein, or any combination of such back-end, middleware, or front-end components.
  • a client and server are generally, but not exclusively, remote from each other and typically interact through a communication network, although the components of the system can be interconnected by any form or medium of digital data communication.
  • Examples of communication networks include, but are not limited to, a local area network (“LAN”), a wide area network (“WAN”), and the Internet.
  • LAN local area network
  • WAN wide area network
  • Internet the Internet
  • Proximal Gap HC Remove false positives caused by nearby misaligned small insertion and deletion events. Reject candidate site if there are ⁇ 3 reads with insertions within an 11-bp window centered on the candidate mutation, or if there are > 3 reads with deletions within the same 11-bp window
  • Triallelic Site HC Reject false positives caused by calling tri-allelic sites where the normal is heterozygous with alleles A/B and MuTect is considering an alternate allele C. Although this is biologically possible, and remains an area for future improvement in mutation detection, calling at these sites generates many false positives and therefore they are currently filtered out by default. However, it may be desirable to review mutations failing only this filter for biological relevance and orthogonal validation and further study the underlying reasons for these false positives.
  • Strand Bias HC Reject false positives caused by context specific sequencing errors where the vast majority of the alternate alleles are observed in a single direction of reads.
  • We perform this test by stratifying the reads by direction and then applying the core detection statistic on the two datasets.

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Abstract

L'invention concerne un système, un procédé et un produit de programme informatique permettant de détecter des variants à partir de données de séquençage. Le procédé consiste à : prévoir des données de séquençage alignées, et appliquer des filtres aux données de séquençage alignées ; utiliser les données filtrées en tant que données d'entrée, et appliquer un premier classificateur pour déterminer la présence d'une altération au-delà d'un seuil due à une erreur de séquençage, et identifier des variants candidats ; faire passer les variants candidats identifiés par des filtres supplémentaires afin d'éliminer les faux positifs ; déterminer, au moyen d'un second classificateur, un état somatique des variants candidats identifiés. L'invention concerne également un appareil, des systèmes, des techniques et des articles correspondants.
PCT/US2013/057128 2012-08-28 2013-08-28 Détection de variants dans des données de séquençage et un étalonnage WO2014036167A1 (fr)

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